The present invention relates to obtaining deoxyribonucleic acid (DNA) from fish spermatogonium in a large scale and, more particularly, to a methodology that entails disrupting fish spermatogonia, followed by a protein separation step and a DNA precipitation step.
DNA is a biopolymer that encodes genetic information and that exists widely in various live organisms. DNA is composed of phosphorous acid, four (4) kinds of bases, deoxyribose, and has been widely used in biochemical experimental materials, cosmetics, medicines, food additives, and etc., due to its intrinsic physicochemical or biological properties.
Squid spermatogonium is a by-product obtained in a large amount from squid food processing industry. After it is captured and its intestines are eliminated, the squid is processed for producing dried squids or dried squid slices. The egg and spermatogonia of squid are processed for use of side dishes in restaurants. The spermatogonium of squid contains a larger amount of DNA than any other materials do and thus, can be used as a good DNA source. Traditionally, DNA has been obtained from spermatogonia of herring or salmon. Korean Patent No. 35973 discloses the use of an anionic surfactant and sodium chloride for producing DNA.
A process also has been disclosed for obtaining DNA in a large scale, either using phenol (U.S. Pat. No. 3,838,148) or using an anionic surfactant with a highly concentrated sodium chloride (Korean Patent No. 35973). However, these methods generate a large amount of pollutants during the process and thus, result in a high cost for treating the pollutants. Therefore, a new process for obtaining DNA from fish spermatogonium without generating of pollutants, preferably by-products produced from the process may be used for manure, has been desired in this field.
Therefore, the present inventors have studied for a long time to develop a novel and economic process for obtaining DNA from fish spermatogonium without the disadvantages of the prior methods, and have found that DNA can be obtained from fish spermatogonia effectively and economically, by using highly concentrated alkaline solution.
It therefore is an object of the present invention to provide a method for obtaining DNA from fish spermatogonia, avoiding the environmental and other problems of conventional techniques, by using highly concentrated alkaline solution without resort to a phenol which is harmful to humans, SDS (Sodium Dodecyl Sulphate), to an anionic surface active agent or an environmental pollutant, such as sodium chloride, which is harmful to soil. As a result, by-products generated during a DNA obtaining process of the present invention are suitable for use as plant nutrient.
Another object of the present invention is to provide a liquid nitrogenous manure containing by-products generated from the method of the present invention.
These and other objects of the present invention can be achieved via a method for obtaining DNA from fish spermatogonium that comprises:
i) disrupting a fish spermatogonium to produce a milky-white colloid containing DNA;
ii) treating the milky-white colloid with alkaline solution of pH 8 to pH 12 which contains more than 1 M of salts, such as a monovalent salt, to separate DNA from nucleosome; and
iii) precipitating DNA by adding ethanol to the mixture obtained in the step (ii).
The above objects of the present invention are also achieved by providing a method for obtaining DNA from fish spermatogonium, which comprises:
i) disrupting a fish spermatogonium in an alkaline solution of pH 8 to pH 12 which contains more than 1 M of salts, such as monovalent salts;
ii) adding an anhydrous compound to the disrupted spermatogonium mixture obtained in the step (i), to effect acylation reaction;
iii) precipitating DNA by adding ethanol to the acylated spermatogonium mixture.